Project Summary Despite early detection and adjuvant therapy, breast cancer remains the leading cause of cancer mortality in women, largely due to distant, incurable recurrences arising years, or even decades, after treatment of the primary tumor. These recurrent, metastatic tumors arise from the pool of residual local and disseminated tumor cells (DTCs) that survive primary treatment and persist in the host in a presumed dormant state. Indeed, the presence of bone marrow DTCs following treatment is independently associated with a substantially increased risk of recurrence. At present, however, the mechanisms enabling residual tumor cells to maintain dormancy and ultimately recur are poorly understood, and DTC-directed treatment approaches are non-existent. Consequently, the ability to biologically characterize and therapeutically target dormant DTCs would be a transformational new approach to preventing recurrence and the mortality associated with it. Increasingly, treatment for cancer patients involves targeted therapies. In particular, targeted inhibition of HER2 in breast cancer patients in the adjuvant setting represents a clear example in which residual tumor cells can be kept at bay for extended periods of time. Using genetically engineered mouse models that faithfully recapitulate tumor dormancy and recurrence, we have discovered that tumor cells that survive targeted HER2 inhibition exist in a state of cellular dormancy at both local and distant sites, and retain the ability to resume growth and give rise to recurrent tumors for the lifetime of the animal. In addition, our preliminary data indicate that dormant residual tumor cells that survive targeted therapy share multiple features with tumor cells that are dormant by virtue of being exposed to microenvironmental cues in the bone marrow and other sites. We hypothesize that tumor cells surviving targeted therapies exist in a dormant state resembling that induced by microenvironmental cues, that the mechanisms underlying dormancy at local and distant sites are related, and that features of dormancy observed in mouse models are recapitulated in patients. The specific aims of this proposal are to: (1) Determine the functional and phenotypic relationship of tumor dormancy in DTCs surviving targeted therapy or microenvironmental selection pressures; (2) Determine whether similar mechanisms regulate tumor dormancy at local and distant sites; and (3) Determine whether bone marrow DTCs in breast cancer patients in the I-SPY SURMOUNT trial exhibit features of tumor dormancy similar to those in mouse models. These aims will be accomplished using a novel flow cytometry approach to isolate and profile DTCs in mouse models and breast cancer patients, and by functionally evaluating mechanisms regulating dormancy on the survival and recurrence of RTCs at local and distant sites, and in contexts in which dormancy has been induced by targeted therapy or the microenvironment. Results of the proposed studies have the potential to transform treatment options for breast cancer survivors by enabling efforts to target the unique vulnerabilities of dormant DTCs, thereby preventing metastatic breast cancer recurrence.